CN102482681B

CN102482681B - Stress tolerant plants

Info

Publication number: CN102482681B
Application number: CN201080035831.2A
Authority: CN
Inventors: 奈斯托·卡里洛; 玛丽安娜·吉罗; 安娜贝拉·费尔南达·洛德伊罗; 马蒂亚斯·丹尼尔·楚尔布里根
Original assignee: PLANT BIOLOGICAL SCIENIC CO Ltd
Current assignee: PLANT BIOLOGICAL SCIENIC CO Ltd; Plant Bioscience Ltd
Priority date: 2009-08-11
Filing date: 2010-08-11
Publication date: 2014-12-17
Anticipated expiration: 2030-08-11
Also published as: GB0914012D0; AU2010283592B2; DE112010003268T5; US20120204291A1; CA2770550A1; BR112012003093A2; EP2464733A1; AU2010283592A1; WO2011018662A1; ZA201109457B; CL2012000224A1; EA201270263A1; MX2012001819A; CN102482681A; AR077852A1

Abstract

The invention relates to methods for increasing stress tolerance in plants by expressing a nucleic acid encoding a FId polypeptide and a nucleic acid sequence encoding a FNR polypeptide in a plant.

Description

Stress tolerant plants

Invention field

The present invention relates to the method for generation of having the plant of coercing the patience that especially oxidative stress strengthens.The invention still further relates to for this method gene expression construct and there are transgenic plant to the patience of coercing enhancing, such as, by the plant that maybe can be obtained by methods described herein that methods described herein obtain.

Introduction

Adversely affect growth, grow or produce external conditions cause extensive plant responding, the change of the genetic expression such as changed, cellular metabolism and growth velocity and crop yield.Have coercing of two types: biotic is applied by other biological, such as pathogenic agent, and abiotic stress results from excessive or not enough physics or chemical environment, such as arid, salinity, high or low temperature or UV-light.

Environment-stress is the key constraints of plant production rate and crop yield.When vegetable cell is under environment-stress, some chemically different reactive oxygen specieses (ROS) are produced by the partial reduction of molecular oxygen and these can cause oxidative damage or serve as signal.The autoxidation of photosynthetic electron transport chain component causes the formation of superoxide radical and derivative, hydrogen peroxide and hydroxyl.These compounds and various biomolecules (comprising DNA) are reacted, and cause cells arrest and dead (Kim etc. 2008, Vranov á etc. 2002).

But flavodoxin (Fld) is found in bacterium and some marine algas the electron transfer flavoprotein (Zurbriggen etc. do not found in plant, 2007), it can participate in the redox path that some ferredoxins (Fd) rely on effectively, comprise the redox modulating (Tognetti etc. of photosynthesis, nitrogen assimilation and Trx mediation, 2006,2007b).In the microorganism being exposed to oxidation and abiotic stress, Fld level is raised (Singh etc., 2004).When being expressed in plant chloroplast, flavoprotein serves as general antioxidant and prevents dissimilar ROS from being formed in chloroplast(id) (Tognetti etc., 2006).The transgenic plant of gained develop multiple patience (Tognetti etc., 2006 to multiple environment challenge, oxidation reduction cycle oxygenant and xenobiotic; 2007a, PCT/GB2002/004612, described All Files is incorporated to herein by reference).In iron deficiency (iron-starved) cyanobacteria, when it is present in Fld conversion of plant, Fld is by Photosystem I (PSI) reduction (Tognetti etc., 2006).In heterotrophic bacteria, Fld can by pyruvic acid-Fld reductase enzyme and NADPH-Fld reductase enzyme reduction (Blaschkowski etc., 1982).Fld also accumulate in cyanobacteria heterocyst composing type and verified it can participate in the electron transmission (Sandmann etc. of nitrogenase, 1990), but the character of terminal electron donor is unknown, and the induction that is more effective, the specific ferredoxin of heterocyst that can mediate this reaction has made people create query (Razquin etc., 1995) to the effect that dinitrogen fixes period Fld.

In plant and cyanobacteria, ferredoxin-NADP (H) reductase enzyme (FNR) (EC1.18.1.2) is the enzyme be combined with thylakoid, its with physics, the mode of composing type participates in from ferredoxin or Fld to NADP ⁺to form the electron transmission (Carrillo and Ceccarelli, 2003) of NADPH.When there is the strong electron pressure from PSI, this is active directly conflicts with the possibility mediating opposite reaction under light illumination.Therefore, the NADPH of FNR mediation can not occur with any significant speed in vivo to the reduction of ferredoxin (or Fld), and does not also report this activity of discovery at present.But FNR and remaining chain of dissolving separate and its (Carrillo and Ceccarelli, 2003) of promptly catalysis.In fact, solvable FNR is mediating the NADP of thylakoid that be separated, FNR disappearance ⁺almost non-activity (Forti and Bracale, 1984) in photoreduction.In the cyanobacteria species comprising the phycobilisome of catching for light, FNR is made up of three structural domains: participate in the N terminal domains that phycobilisome combines, thereafter be FAD binding domains and NADP (H) binding domains, described FAD binding domains forms the active part (Carrillo and Ceccarelli, 2003) of this enzyme together with NADP (H) binding domains.Alternative initiator codon is positioned the beginning of second structural domain to produce the solvable FNR (Thomas etc., 2006) of two structural domains.Be heterotrophism mode of life when cells switch and need from NADPH to Fd or when Fld transmits the ability of electronics, preferably using this inner Met (Thomas etc., 2006).Provide the diagram describing this theoretical model in FIG.Described enzyme is all found that there is in all cyanobacterias and photosynthetic eukaryotic cell.But other enzymes with similar specificity different physiological action are at some non-photosynthetic plant tissues, described at mammalian mitochondria and in some bacteriums.Cyanobacteria FNR is well characterized (Sancho, 1987, Schluchter 1992).In addition, the petH gene of coding FNR clones (Fillat etc., 1993) from some cyanobacteria strains.The existence of detection of active FNR can be measured by diaphorase activity as described below.

Therefore knownly bacterium Fld is attached to the reduction that can compensate Fd level in tobacco chloroplast, produces the patience that oxygenant and extensive bad stress conditions are strengthened.Target of the present invention is by ensureing to maintain the stress tolerance that Fld improves plant in reductive condition.

Summary of the invention

In one aspect, the present invention relates to the method for the plant for generation of the stress tolerance with reinforcement, described method is included in plant expresses the nucleotide sequence of coding flavodoxin polypeptide and the nucleotide sequence of encoding ferredoxin NADP (H) reductase enzyme polypeptide.Plant that is that obtain by this method or that can obtain by this method also within the scope of the invention.The invention further relates to the nucleic acid construct comprising gene order, wherein said gene order comprises the nucleotide sequence of coding cyanobacteria FNR and chloroplast targeted sequence.In one aspect of the method, the present invention relates to such nucleic acid construct, described nucleic acid construct comprises the nucleotide sequence of coding flavodoxin polypeptide (Fld) and the nucleotide sequence of encoding ferredoxin NADP (H) reductase enzyme (FNR) polypeptide.In one aspect of the method, the present invention relates to the transgenic plant of the stress tolerance with reinforcement, the described nucleotide sequence of Expressed in Transgenic Plant coding flavodoxin polypeptide and the nucleotide sequence of encoding ferredoxin NADP (H) reductase enzyme polypeptide.In another aspect, the present invention relates to for reducing the method that in plant, ROS measures in the response of coercing, described method is included in plant expresses flavodoxin polypeptide and ferredoxin NADP (H) reductase enzyme polypeptide.

Accompanying drawing

The present invention is illustrated further in non-limitative drawings.

Fig. 1. expressing the electron path from the proposal in two transformant of Fld and FNR of cyanobacteria.Under normal growing conditions (upper figure), ferredoxin (Fd) and Fld both can mediate the electron transmission in production path, are likely preferred according to efficiency Fd.Under coercing (figure below), Fd level declines and Fld has taken over the photosynthetic electron transfer arriving NADP, and the NADPH that simultaneously solvable FNR uses part to be formed is reduced to keep Fld, prevents formation ROS and terminates benign cycle.

Fig. 2. the FNR accumulation in the blade of wild-type (PH) and transformant tobacco.Be equivalent to the leaf extract from independent transformation plant in 6 week age (pnn) of 17 μ g albumen by SDS-PAGE classification and for the antiserum(antisera) immunodetection FNR used for fish raw meat cyanobacteria (Anabaena) reductase enzyme on trace to nitrocellulose membrane.

Fig. 3. from Subcellular Localization and the glue myocardium yellow enzyme activity of the FNR of rotaring gene tobacco plant.A), after the complete excision be separated from wild-type with pFNR plant at osmotic shock, thylakoid is separated with matrix.Be equivalent to the chlorophyllous sample of 4 μ g be separated by SDS-PAGE and determined the existence of FNR by immunoblotting assay.B) be equivalent to the matrix from wild-type and pFNR plant of the total soluble protein of 15 μ g, by non denatured electrophoretic separation, diaphorase activity dyeed.

Fig. 4. FNR and the Fld expression level in X4 plant generations.Be equivalent to the total soluble protein of 8 μ g from 6 week age tobacco plant leaf extract by SDS-PAGE classification and trace on nitrocellulose membrane, for using for the sero-fast immunodetection of fish raw meat cyanobacteria FNR and Fld.

Fig. 5. methyl viologen (MV) expresses the effect of c4 plant leaf discs to FNR/Fld.By from 6 week age tobacco plant leaf dish to be placed on 20 μMs of MV and with 600 μm of ol quanta m ^-2s ^-1irradiate.A) photo taken after incubation 7h.B) after with MV process leaf dish, Ion leakage is assessed by the increase of measuring media Relative electro-conductivity.C) after 7h MV process, Chlorophylls and Carotenoids is determined.

Fig. 6 .MV is to the effect of whole tobacco plant.Surrounding plant in age is transferred to hydroponic system.After being exposed to 100 μMs of MV 24h, the photo of blade is taken under growth room's condition.

Fig. 7 .A) detect lipid peroxide.By from 6 week age plant leaf dish be placed on 10 μMs of MV or (bar of the right hand) waterborne and with 700 μm of ol quanta m in 3h ^-2s ^-1irradiate.Each value is the mean+SD of four sample repeated measures.B) APX expressing the leaf extract of the leaf dish of plant from FNR/Fld is active.By from 6 week age tobacco plant leaf dish to be placed on 20 μMs of MV and with 600 μm of ol quanta m ^-2s ^-1irradiate.1.5 and 3h incubation after collected specimens.

Fig. 8. comprise (in-frame) in the frame between coding pea FNR transit peptides and the sequence of flavodoxin gene and merge the diagram of the binary vector pCAMBIA 2200 of fragment.The box being inserted in the Eco RI site of pCAMBIA 2200 is structured in pDH51 in advance.This Eco RI fragment comprises CaMV 35S promoter, flavodoxin mosaic gene and CaMV35S polyadenylation signal.

Fig. 9. the diagram of binary vector pCAMBIA 2200 of the frame endomixis fragment between the sequence comprising two C terminal domains of coding pea FNR transit peptides and fish raw meat cyanobacteria FNR gene.The box being inserted in the Eco RI site of pCAMBIA2200 is structured in pDH51 in advance.This Eco RI fragment comprises CaMV 35S promoter, FNR mosaic gene and CaMV35S polyadenylation signal.

Figure 10. be derived from the binary vector pBinary-BRACT B1 of MultiSite Gateway, 4-ubi-FNR/B2, the diagram of 3-actin-Fld, described carrier comprise coding pea FNR transit peptides and fish raw meat cyanobacteria FNR gene two C terminal domains sequence between, coding pea FNR transit peptides and from Fld (TP-Fld) gene of fish raw meat cyanobacteria PCC7119 sequence between frame endomixis.In co-expression carrier, no polyadenylation signal and ubi and actin promoter are respectively in the both sides of TP-FNR and TP-Fld construct.First these constructs are cloned in the suitable donor vehicle of pDONR221 carrier families by locus specificity BP recombining reaction.Enter (entry) of gained clones the double base T-DNA MultiSite Gateway object carrier and pDEST-BRACT R1 that participate in again simultaneously and customize, 4-ubi/R2, two LR Site-specific recombinase of 3-actin, produce the cloning by expression pBinary-BRACT B1 comprising two goal gene, 4-ubi-FNR/B2,3-actin-Fld.Described construct reacts (Invitrogen, http://www.invitrogen.com) to BP and the LR Site-specific recombinase of the Strategies For The Cloning in binary vector based on MultiSite Gateway technology.

Hyg: selective marker (hygromycin (hygromicin)); LB: left border; Nos: rouge alkali synthetase; RB: right side boundary; TP: transit peptides; Ubi: ubiquitin.

Figure 11. for the structure of the binary vector of coexpression Fld and FNR polypeptide in plant.Schematic diagram display is used for the pBinary-BRACT B1 of coexpression FNR and Fld in plant, the structure of 4-ubi-FNR/B2,3-actin-Fld binary vector.Both sides have PCR primer (attB1-FNR-attB4 and attB2-Fld-attB3 of coding FNR and Fld to the sequence of the chimeric fusion of chloroplast targeted transit peptides (TP) of attB Site-specific recombinase sequence, respectively) (pDONR21 P1-P4 and pDONR p2-P3 is substrate in the BP recombining reaction carried out respectively) with appropriate donor carrier.The pENTR221 L1-L4-FNR of gained and pENTR221 L2-L3-Fld enters the double base T-DNA MultiSite Gateway object carrier and pDEST-BRACT R1 that clone participates in again simultaneously and customize, 4-ubi/R2, two LR Site-specific recombinase of 3-actin, generation is included in the cloning by expression of two goal gene under constitutive promoter control.Described process (Invitrogen, http://www.invitrogen.com) is carried out according to experimental program, operation instruction and nomenclature that manufacturers advises.CcdB: for the gene of the Solid phase of carrier; LB: left border; Nos: rouge alkali synthetase; RB: right side boundary; TP: transit peptides; Ubi: ubiquitin.

Figure 12. barley is coerced.Methyl viologen (MV) expresses the effect of the leaf bar (strip) of heterozygosis barley plants to FNR/Fld.From growth soil 6 week age barley plants blade cut the leaf bar of 10-15mm length.Then in the dark leaf bar incubation in 50 μMs of MV and 0.05% Tween-20 is diffused in tissue to allow MV for 30 minutes at 20 DEG C.Then described leaf bar is placed in plastic pallet and makes in paraxial side direction towards 450 μm of ol quanta m ^-2s ^-1light source.Contrast is remained on comprise in the distilled water of 0.05%Tween-20.Illumination later evaluation A at 7.5h) chlorophyll and B) content of carotenoid.FNR (1x): heterozygosis has the transgene barley of FNR.Fld (1x): heterozygosis has the transgene barley of Fld.FNR/Fld (1x): heterozygosis has FNR and Fld transgene barley.WT: wild-type barley.

Describe in detail

Present invention will be further described now.In the following paragraphs, different aspect of the present invention is limited in more detail.The each side of such restriction can be combined with any other aspect, unless clearly illustrated in contrast.Especially, any be shown as preferred or favourable feature can be shown as any other preferred or favourable integrate features.

Unless otherwise indicated, enforcement of the present invention is by the routine techniques of phytology, microbiology, tissue culture, molecular biology, chemistry, biological chemistry and the recombinant DNA technology in employing art technology.Such technology obtains full-time instruction in the literature.

As mentioned above, known bacterium flavodoxin (Fld) is attached to the decline that can compensate Fd level in tobacco chloroplast, produces the patience that oxygenant and extensive bad stress conditions are increased.The present inventor has shockingly found second gene with Fld reducing activity deriving from bacterium to introduce the stress tolerance that can improve this plant in the plant of expressing bacterium Fld.Do not wish that the present inventor believes that this is owing to being maintained in reductive condition by Fld by theoretical restriction.As shown in Example, the present inventor has used to have and has derived from cyanobacteria and the construct of the nucleotide sequence of ferredoxin NADP (H) reductase enzyme (FNR) polypeptide of encoding chloroplast target and express described bacterial gene in the plant of expressing chloroplast targeted Fld.

Therefore, in one aspect, the present invention relates to the method for the plant for generation of the stress tolerance with enhancing, described method is included in plant the nucleotide sequence of nucleotide sequence and the coding FNR polypeptide of expressing coding Fld polypeptide.The expression of these sequences according to the present invention in plant can be realized by different modes as described herein.

In the first embodiment, described method is included in the nucleic acid construct of expressing as described herein in plant and instructing Fld polypeptide and FNR coexpression.Therefore, the coexpression of two genes can be instructed in the plant of this construct transforming with good grounds different aspect of the present invention according to the single construct of different embodiments as detailed in this article.The transgenic plant of gained produce Fld and FNR polypeptide.In this approach, produce with coexpression construct conversion of plant and select the genetically modified stable heterozygote plant of expression two kinds.

Below describe the construct that may be used for this method in detail.

Described nucleic acid construct comprises the nucleotide sequence of coding Fld polypeptide and the nucleotide sequence of coding FNR polypeptide.Preferably, Fld and FNR sequence derives from bacterium.

In one embodiment, the nucleotide sequence of Fld polypeptide of encoding derives from cyanobacteria and flavodoxin polypeptide is cyanobacteria flavodoxin.Alternatively, the nucleotide sequence of coding Fld polypeptide derives from heterotrophic bacterium.Cyanobacteria can be selected from Crocosphaera, blue Pseudomonas (Cyanobium), blue silk Pseudomonas (Cyanothece), micro-capsule cyanobacteria belongs to (Microcystis), Synechococcus belongs to (Synechococcus), collection born of the same parents cyanobacteria belongs to (Synechocystis), Thermosynechococcus, microtriche cyanobacteria belongs to (Microchaetaceae), Nostocaceae, sheath silk cyanobacteria belongs to (Lyngbya), spiral cyanobacteria belongs to (Spirulina) or restraints darkish blue bacterium genus (Trichodesmium).Preferred genus comprises Synechococcus and belongs to (Synechococcus), Fremyella, single discrimination cyanobacteria belongs to (Tolypothrix), fish raw meat cyanobacteria belongs to (Anabaena), necklace cyanobacteria belongs to (Anabaenopsis), blue beam silk cyanobacteria belongs to (Aphanizomenon), pipe chain cyanobacteria belongs to (Aulosira), Cylindrospermopsis, cylinder spore cyanobacteria belongs to (Cylindrospermum), Loefgrenia, joint ball cyanobacteria belongs to (Nodularia), read ball cyanobacteria and belong to (Nostoc) or lumen cyanobacteria genus (Wollea).Preferably, described genus be fish raw meat cyanobacteria belong to and cyanobacteria to be fish raw meat cyanobacteria belong to PCC7119 (Fillat etc. 1990).

In one embodiment, Fld sequence has the nucleotide sequence of the sequence be selected from as shown in table 1 below.In one embodiment, FNR sequence has the nucleotide sequence of the sequence be selected from as shown below in table 2.

Table 1

Table 2.

In another embodiment, the nucleotide sequence of coding cyanobacteria Fld comprises SEQ ID NO.1.Corresponding aminoacid sequence is presented in SEQ ID NO.6.The variant of SEQ ID NO.1 or SEQ ID NO.6 also within the scope of the invention.Variant retains the biologic activity of described protein.

Further, the present invention relates to the method for generation of the plant of the stress-tolerance with reinforcement and the method increasing stress tolerance in plants, described method is included in plant the nucleotide sequence of expressing coding FNR polypeptide.In plant according to the present invention, express these sequences to be realized by modes different as described in this article.In another embodiment, described FNR polypeptide is by the polypeptide of SEQ ID NO:8 or 9 representative, or is shown in polypeptide or its cyanobacteria homologue of table 2.As shown in Example, the present inventor has used with deriving from cyanobacteria and the construct of the nucleotide sequence of ferredoxin NADP (H) reductase enzyme (FNR) polypeptide of encoding chloroplast target and express described bacterial gene in plant.

In one embodiment, the nucleotide sequence of FNR polypeptide of encoding derives from cyanobacteria and described FNR polypeptide is cyanobacteria FNR.Described cyanobacteria can be the bacterium comprising phycobilisome, such as be selected from Crocosphaera, blue Pseudomonas (Cyanobium), blue silk Pseudomonas (Cyanothece), micro-capsule cyanobacteria belongs to (Microcystis), Synechococcus belongs to (Synechococcus), collection born of the same parents cyanobacteria belongs to (Synechocystis), Thermosynechococcus, microtriche cyanobacteria belongs to (Microchaetaceae), Nostocaceae, sheath silk cyanobacteria belongs to (Lyngbya), spiral cyanobacteria belongs to (Spirulina) or restraints darkish blue bacterium genus (Trichodesmium).Preferred genus comprises Synechococcus and belongs to (Synechococcus), Fremyella, single discrimination cyanobacteria belongs to (Tolypothrix), fish raw meat cyanobacteria belongs to (Anabaena), necklace cyanobacteria belongs to (Anabaenopsis), blue beam silk cyanobacteria belongs to (Aphanizomenon), pipe chain cyanobacteria belongs to (Aulosira), Cylindrospermopsis, cylinder spore cyanobacteria belongs to (Cylindrospermum), Loefgrenia, joint ball cyanobacteria belongs to (Nodularia), read ball cyanobacteria and belong to (Nostoc) or lumen cyanobacteria genus (Wollea).In one embodiment, genus is that fish raw meat cyanobacteria belongs to and cyanobacteria is fish raw meat cyanobacteria belongs to PCC7119 (Fillat etc. 1990).Preferably, described sequence comprises the sequence that coding C holds two domain region, but does not comprise the region of coding phycobilisome binding domains.Such as, the nucleotide sequence of coding cyanobacteria FNR comprises SEQ ID NO.3.Corresponding aminoacid sequence is presented in SEQ ID NO.9.The variant of SEQ ID NO.3 or SEQ ID NO.9 also within the scope of the invention.Variant retains the biologic activity of described protein.

Described construct can be heterologous gene construct, and wherein Fld and FNR coding nucleic acid derives from different biologies.In another embodiment, Fld and FNR coding nucleic acid both derives from identical biology, such as cyanobacteria.In one embodiment, two nucleotide sequences all derive from fish raw meat cyanobacteria.Such as, described construct can comprise as the sequence as shown in SEQ ID 1 and 3 or its functional variant.

In preferred embodiments, construct described above also comprises at least two chloroplast targeted sequences (encoding transit peptides) and is targeted to chloroplast(id) to make each polypeptide.According to the present invention, be suitable by pg polypeptide to any sequence of chloroplast(id).Example is presented in the table 2 being incorporated to PCT/GB2002/004612 herein by reference.Such as, target sequence can derive from pea FNR.

Therefore, in a preferred embodiment of the present invention, described construct can comprise one, preferably as the sequence as shown in SEQ ID 2 and 4 or its functional variant.

Construct described above instructs the nucleotide sequence of coding Fld and FNR polypeptide from single construct coexpression.Preferably, described construct comprises the polypeptide of at least two chloroplast targeted sequences thus encoding chloroplast target.As embodiment, according to fusion constructs of the present invention, Figure 11 illustrates this construct and how to make (also seeing embodiment) in Figure 10 display.

Construct described above also within the scope of the invention.In other words, the present invention relates to such nucleic acid construct, described nucleic acid construct comprises the nucleotide sequence of coding Fld polypeptide and the nucleotide sequence of coding FNR polypeptide.The multiple embodiments of construct and preferred sequence is set forth in.

In any construct as herein described, the wild-type sequence of coding Fld or FNR polypeptide is preferred, but also can use mutant/variant sequence thereof or fragment, as long as such sequence encoding and wild-type sequence have the polypeptide of same biologic activity.The sequence variation of wild-type sequence comprises the reticent base not causing the aminoacid sequence of coding to change and changes and/or affect the base change that aminoacid sequence does not still affect polypeptide biologic activity.Change can be conservative amino acid replacement, namely replaces an amino-acid residue when two residues have similar quality.Therefore, retained the biologic activity of wild type peptide by the variant/mutant polypeptide of such sequence encoding and given stress tolerance.Such as, the sequence variation (as shown in SEQ ID NO.3) that FNR nucleotides sequence is listed in following position does not show the activity of polypeptide as described in impact: 535:A/G; Asn (AAC)/Asp (GAC), 703:A/G; Met (ATG)/Val (GTG), 763:C/G; Gln (CAA)/Glu (GAA).Therefore, the variant of these alternative Nucleotide/amino acid whose FNR nucleotide sequence/aminoacid sequence is comprised in the scope of embodiment of the present invention.

The nucleic acid used according to the present invention can be double-strand or strand, cDNA, genomic dna or RNA.Any sequence as herein described, the sequence of such as FNR with Fld gene can be separated the sequence from plant, bacterium or artificial synthesized sequence.Depend on design, nucleic acid can be all or part of synthesis.Understanding when nucleic acid according to the present invention comprises RNA, is mentioned that shown sequence should be counted as the equivalent mentioning RNA by technician, and wherein U replaces T.

In addition, the present invention relates to the homologue of FNR or FLD polypeptide and its purposes in method of the present invention, construct and carrier.The homologue of FNR or FLD polypeptide with represented by SEQ ID NO:8 to 10 or SEQ ID NO:6 or 7 respectively and/or the amino acid that represented by its straight homologues be shown in table 2 and table 1 and paralog thing respectively have, with the preferable increased, overall sequence iden below: at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.Use Needleman Wunsch algorithm (the GCG Wisconsin Package in overall comparison algorithm such as GAP program, Accelrys), preferably use default parameter and preferably use the sequence of mature protein (namely not considering secretion signal or transit peptides) to determine overall sequence iden.

According to the further embodiment of the present invention, provide and use the method for the following, and comprise the construct of the following, host cell, plant and carrier:

A) nucleic acid molecule be separated, described nucleic acid molecule is selected from:

I () is by SEQ ID NO:1 or 2 or the encoding list nucleic acid in those representatives of the homologue of table 1;

(ii) by SEQ ID NO:1 or 2 or the encoding list complement in the nucleic acid of those representatives of the homologue of table 1;

(iii) coding is as by the nucleic acid in the polypeptide of those representatives of table 1 of any one or list in SEQ ID NO:6 or 7, preferably as the result of degenerate, the nucleic acid of described separation can derive from as further preferably given the stress tolerance of the reinforcement relative to control plant by SEQ ID NO:6 or 7 (in any one) or list in peptide sequences of those representatives of table 1;

(iv) have in the nucleotide sequence of the homologue (preferably the homologue of SEQ ID NO:1 or 2) of table 1 with any one or the encoding list in the nucleotide sequence of SEQ ID NO:1 or 2, with the preferable increased, the nucleic acid of following sequence iden: at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and described nucleic acid further preferably gives the stress tolerance of the reinforcement relative to control plant,

V () also preferably gives the nucleic acid molecule of the stress tolerance of the reinforcement relative to control plant with the making nucleic acid molecular hybridization of (i) to (iv) under strict hybridization conditions;

(vi) nucleic acid of coding FLD polypeptide, described FLD polypeptide has with any other aminoacid sequence in the aminoacid sequence represented by SEQ ID NO:6 or 7 (in any one) and table 1, with the preferable increased, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the sequence iden of 98% or 99%, and preferably give the stress tolerance of the enhancing relative to control plant,

And

B) nucleic acid molecule be separated, described nucleic acid molecule is selected:

I () is by SEQ ID NO:3 or 4 or the encoding list nucleic acid in those representatives of the homologue of table 2;

(ii) by SEQ ID NO:3 or 4 or the encoding list complement in the nucleic acid of those representatives of the homologue of table 2;

(iii) coding is as by the nucleic acid in the polypeptide of those representatives of table 2 of any one or list in SEQ ID NO:8 or 9, preferably as the result of degenerate, the nucleic acid of described separation can derive from as further preferably given the stress tolerance of the reinforcement relative to control plant by SEQ ID NO:8 or 9 (in any one) or list in peptide sequences of those representatives of table 2;

(iv) have in the nucleotide sequence of the homologue (preferably the homologue of SEQ ID NO:3 or 4) of table 2 with any one or the encoding list in the nucleotide sequence of SEQ ID NO:3 or 4, with the preferable increased, the nucleic acid of following sequence iden: at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and described nucleic acid further preferably gives the stress tolerance of the reinforcement relative to control plant,

(vi) nucleic acid of coding FLD polypeptide, described FLD polypeptide has with any other aminoacid sequence in the aminoacid sequence represented by SEQ ID NO:8 or 9 (in any one) and table 2, with the preferable increased, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the sequence iden of 98% or 99%, and preferably combine the stress tolerance that the FLD polypeptide be present in as described herein in plant gives the enhancing relative to control plant.

In further embodiment, provide and use the method for the following, and comprise the construct of nucleic acid molecule of the separation being selected from the following, host cell, plant and carrier:

Preferably, any one whole peptide sequence is determined any comparison of sequence iden in SEQ ID NO:6 to 9 to peptide sequence, or in SEQ ID NO:1 to 4 nucleotide sequence of any one whole coding region on nucleotide sequence determined to any comparison of sequence iden.Such as, for determining the sequence iden of the peptide sequence of peptide sequence and SEQ ID NO:8, aligned sequences in the whole length of SEQ ID NO:8.

Control plant is such plant, and it does not comprise restructuring FLD with FNR of the present invention but identical as much as possible and by with the mode process identical with plant of the present invention in every other.

In one embodiment, the functional variant of FLD or FNR polypeptide is such polypeptide, described polypeptide respectively with the FLD such as represented by the sequence of SEQ ID NO:6 or 7, or there is substantially identical biologic activity as the FNR that represented by the sequence of SEQ ID NO:8 or 9.In another embodiment, functional variant be polypeptide homolog as defined herein or encoded by the nucleic acid sequence homologs that limits above those.

All nucleic acid constructs as herein described can also comprise adjustment sequence.Therefore, nucleotide sequence as herein described can under regulating the operation of sequence to control, and described adjustment sequence can control the genetic expression in plant.Adjustment sequence can be the promoter sequence of the genetic expression driven in construct.Such as, the promoter expression nucleotide sequence driving process LAN can be used.According to the present invention, process LAN refers to the horizontal expression transgenosis higher than the expression of the endogenous counterpart driven by its endogenesis promoter (plant FNR or Fd).Such as, can use strong promoter, any promotor of the expression that such as cauliflower mosaic virus promoter (CaMV35S), rice actin promoters or maize ubiquitin promoter or generation are strengthened implements process LAN.Alternatively, can transcribe or translational enhancer or activate expression that son realizes strengthening or strengthening and enhanser can be attached in gene thus Enhanced expressing further by using.In addition, can use can inducible expression, wherein express by promoters driven (the membrane protein gene CaPIMPI of such as pepper pathogen-inducible or comprise the promotor of dehydration response element (dehydration-responsive element) (DRE) induced by environmental stress conditions, can be coerced by water, the promotor (Dezar etc. of Sunflower Receptacle HD-Zip protein gene Hahb4 of high salt concentration and ABA induction,, or chemical inducible promoters (such as steroid or ethanol inducible promoter system) 2005).Such promotor is such as stated in the art in Pastori (2002).For technician, other suitable promotors and can inducible system also be known.

If technician is by understanding, described construct also can comprise the selected marker helping and select transformant, such as gives the mark of the resistance to microbiotic such as kantlex.

As detailed above, in an embodiment of the inventive method, single construct is used to instruct the coexpression of Fld and FNr nucleic acid sequence encoding.

In another embodiment, the method for plant for generation of the stress tolerance with reinforcement comprises:

A) express nucleic acid construct in plant, described construct comprises the sequence of coding Fld polypeptide,

B) express nucleic acid construct, described construct comprises the sequence of FNR polypeptide as described herein of encoding,

C) the first and second plant hybridizations are made, and

D) generation expresses FNR and Fld and the plant of both isozygotying for FNR and Fld.

According to the first step of described method, transform the first plant with the nucleic acid construct of the sequence comprising coding flavodoxin polypeptide.Such construct is stated in (2006) such as Tognetti and PCT/GB2002/004612, is incorporated to all by reference herein both it.Preferred construct comprises and is derived from cyanobacteria, preferably fish raw meat cyanobacteria, the most preferably sequence of fish raw meat cyanobacteria PCC7119.Described construct preferably comprises the transit peptides of targeting proteins to chloroplast(id).Suitable construct is also shown in Fig. 8.In preferred embodiments, described construct also comprises chloroplast targeted sequence, such as, be derived from the sequence of pea.Polypeptide is targeted to chloroplast(id) by described transit peptides.In preferred embodiments, described construct comprises the sequence as display in SEQ ID NO.1 or 2.Obtain and express the genetically modified stable conversion body of Fld.

In second step, transform the second plant with the nucleic acid construct of the sequence comprising FNR polypeptide as described herein of encoding.Produce express FNR genetically modified, be the stable transformant of isozygotying concerning transgenosis.

Below describe the nucleotide sequence and the nucleic acid construct that may be used for the different embodiments of context of methods that comprise coding FNR polypeptide in detail.The nucleotide sequence of coding FNR preferably derives from bacterium and most preferably derives from cyanobacteria.

Described cyanobacteria can be the bacterium comprising phycobilisome, such as be selected from Crocosphaera, blue Pseudomonas (Cyanobium), blue silk Pseudomonas (Cyanothece), micro-capsule cyanobacteria belongs to (Microcystis), Synechococcus belongs to (Synechococcus), collection born of the same parents cyanobacteria belongs to (Synechocystis), Thermosynechococcus, microtriche cyanobacteria belongs to (Microchaetaceae), Nostocaceae, sheath silk cyanobacteria belongs to (Lyngbya), spiral cyanobacteria belongs to (Spirulina) or restraints darkish blue bacterium genus (Trichodesmium).Preferred genus comprises Synechococcus and belongs to (Synechococcus), Fremyella, single discrimination cyanobacteria belongs to (Tolypothrix), fish raw meat cyanobacteria belongs to (Anabaena), necklace cyanobacteria belongs to (Anabaenopsis), blue beam silk cyanobacteria belongs to (Aphanizomenon), pipe chain cyanobacteria belongs to (Aulosira), Cylindrospermopsis, cylinder spore cyanobacteria belongs to (Cylindrospermum), Loefgrenia, joint ball cyanobacteria belongs to (Nodularia), read ball cyanobacteria and belong to (Nostoc) or lumen cyanobacteria genus (Wollea).

As shown in Example, the FNR gene from fish raw meat cyanobacteria PCC7119 can be used.Disappearance is fallen the 3rd structural domain and is introduced in tobacco by the mosaic gene of gained.Therefore, in one embodiment, described genus is fish raw meat cyanobacteria.Preferably, but described sequence comprises coding C and holds the sequence of two domain region do not comprise the region of coding phycobilisome binding domains.The full length sequence of FNR is presented in SEQ ID NO.5.Such as, described construct can comprise SEQ ID NO.3.Described construct preferably can comprise coding by the sequence of described targeting proteins to the transit peptides of chloroplast(id).Transit peptides is chloroplast targeted peptide.This is preferably derived from plant FNR, such as pea.Such as, described construct can comprise SEQ ID NO.4.As embodiment, Fig. 9 display is according to construct of the present invention.

In the third step, the first stable transformant and the stable transformant of the second hybridize to produce the stable progeny plants of isozygotying expressing FNR and Fld.If technician is by understanding, be hemizygous " hybrid " by generation to each gene by Fld plant and FNR plant hybridization.The plant of gained needs selfing then to select offspring to find that two homozygote-is namely all the plant of isozygotying for two transgenosiss.Technician also will understand polyploid to be needed more than a step " selfing ".Therefore, produce express FNR and Fld and the step of the plant of both isozygotying for FNR and Fld comprises generation passes through steps d) offspring of plant that obtains and select for two transgenosiss to be all the plant of isozygotying.As shown in Example, express after compatriot hybridize making FNR plant and Fld, two homozygote plant selected and demonstrate relative to simple close Fld plant to causing the better patience of the oxidation reduction cycle compounds methyl purpurine (MV) of oxidative stress.

A) express nucleic acid construct in plant, described construct comprises the sequence of flavodoxin polypeptide in coded plant or FNR polypeptide,

B) described plant is transformed with the nucleic acid construct of the sequence comprising encode respectively flavodoxin polypeptide or FNR polypeptide, to produce the stable homozygote plant of expressing FNR and Fld.

According to this embodiment, produce single transformant and again transform this single transformant to produce the stable homozygote plant of expressing FNR and Fld with comprising digenic nucleic acid construct.Then stable homozygote plant is selected.

Technician will understand, and use selected marker help to be promoted to select double-mutant to different construct.

The construct that can be used in this embodiment is also being described above.

In another aspect, the present invention relates to such nucleic acid construct, described nucleic acid construct comprises the nucleotide sequence of coding cyanobacteria FNR and chloroplast targeted sequence.Such construct and different embodiments are being described above.

In another aspect, the present invention relates to such carrier, described carrier comprises construct as herein described.Described carrier is preferably suitable for conversion of plant and operable carrier is known to technician.The invention still further relates to the plant host cell comprising construct as herein described or carrier.

The present invention also comprises such host cell, described host cell comprises the recombinant nucleic acid of coding flavodoxin polypeptide and the recombinant nucleic acid sequence of encoding ferredoxin NADP (H) reductase enzyme polypeptide, described two nucleotide sequences all as above limit.Host cell of the present invention can be any cell being selected from the group be made up of the following: bacterial cell, such as intestinal bacteria or Agrobacterium (Agrobacterium) Species Cell, yeast cell, fungal cell, marine alga or cyanobacteria cell, or vegetable cell.In further embodiment, the present invention relates to the construct of the present invention be included in transgenic plant cells.

In another embodiment, vegetable cell of the present invention is non-propagated cell, such as this cell can not be used for using the whole strain plant of standard cell culture techniques from then on cell regeneration generally, and standard cell culture techniques phalangeal cell cultural method still gets rid of cell in vitro core, organoid or chromosome transfer method.

For the present invention, " genetically modified ", " transgenosis " or " restructuring " mean and relate to, such as, nucleotide sequence, expression cassette, gene construct or comprise the carrier of nucleotide sequence or transform the biology of with good grounds nucleotide sequence of the present invention, expression cassette or carrier, all that builds and is produced by such recombination method, wherein

The nucleotide sequence of a albumen that () coding is useful in the method for the invention, or

B () can operate with nucleotide sequence according to the present invention the genetic control sequences be connected, such as promotor, or

C () a) and b)

Uncertainly be arranged in its natural genotypic environment or modified by recombination method, modification may adopt following form, such as, displacement, add, disappearance, inversion or insert one or more nucleotide residue.Natural genotypic environment is understood to mean the existence in genome natural in primordial plant or chromosome position or genomic library.When genomic library, the natural genetic environment of nucleotide sequence is preferably retained at least partly.Described environment is at least side of nucleotide sequence both sides and have at least 50bp, preferably at least 500bp, especially preferably at least 1000bp, most preferably the sequence length of at least 5000bp.The naturally occurring combination of the nucleotide sequence of the natural promoter of naturally occurring expression cassette-such as nucleotide sequence and the useful in the method for the invention polypeptide of corresponding coding, as mentioned above-and when this expression cassette by (" artificial ") method of non-natural, synthesis, such as such as modify by mutagenic treatment, become transgene expression cassette.Suitable method is described in such as US 5,565,350 or WO 00/15815.

Therefore for the present invention, transgenic plant are understood to mean, and as above, for the nucleic acid of method of the present invention not at its natural place in described Plant Genome, described nucleic acid may homology or heterogenous expression.But, as mentioned, transgenosis also refers to, although according to the nucleic acid of different embodiments of the present invention on its natural place in Plant Genome, but modified relative to sequence described in native sequences, and/or the adjustment sequence of native sequences is modified.Preferably transgenosis is interpreted as that the non-natural location presentation referred in genome is according to nucleic acid of the present invention, namely homology or the preferably heterogenous expression of nucleic acid occur.Preferred transgenic plant are mentioned in this article.

For increasing the stress response of plant or patience method also within the scope of the invention, described method is included in plant expresses the nucleotide sequence of coding flavodoxin polypeptide and the nucleotide sequence of encoding ferredoxin NADP (H) reductase enzyme polypeptide.The method uses different construct as herein described and step thus produces stress tolerant plants.Compared to wild-type/control plant and the plant of but not expressing the nucleotide sequence of encoding ferredoxin NADP (H) reductase enzyme polypeptide compared to the nucleotide sequence of only expressing coding flavodoxin polypeptide, stress response is increased.Stress response can increase at least 2 to 10 times or more.

In another aspect, the present invention relates to the transgenic plant that maybe can be obtained by methods described herein obtained by methods described herein.In another aspect, the present invention relates to the transgenic plant of expressing construct described herein.The invention still further relates to the transgenic plant of the stress tolerance with enhancing, the described nucleic acid of Expressed in Transgenic Plant coding flavodoxin polypeptide and the nucleic acid of encoding ferredoxin NADP (H) reductase enzyme polypeptide.

According to the nucleotide sequence of expression of plants coding FNR polypeptide of the present invention, such as comprise as the sequence as shown in SEQ ID NO.8 or 9 or its functional variant, and express the nucleotide sequence of coding Fld polypeptide, be such as included in SEQ ID NO.5, the sequence shown in 6 or 7 or its functional variant.

The present invention also extends to the part gathered in the crops of plant such as, but not limited to seed, leaf, fruit, flower, stem, root, rhizome, stem tuber and napiform root, the described recombinant nucleic acid gathered in the crops part and comprise coding FNR polypeptide, preferably also comprises the recombinant nucleic acid of coding flavodoxin polypeptide.In addition the present invention relates to the product deriving from the part gathered in the crops preferably being directed to such plant, such as dry agglomerate or powder, oil, fat and lipid acid, starch or protein.

In one embodiment, seed of the present invention comprises construct of the present invention or carrier of the present invention.In further embodiment, seed of the present invention is pure breeding for construct of the present invention or carrier.Described seed comprises the recombinant nucleic acid of coding FNR polypeptide and comprises the recombinant nucleic acid of coding flavodoxin polypeptide in another embodiment, both as disclosed herein, and the stress tolerance that the display of described seed increases.

The present invention also comprises the method for generation of product, described method comprise a) plant plant of the present invention and b) from or the seed that comprises these plants by plant of the present invention or part produce described product.In further embodiment, described method comprises step and a) plants plant of the present invention, b) removes from described plant and can gather in the crops as defined above partly and c) certainly or by part of gathering in the crops of the present invention produce described product.

The place that can grow plant produces product, or the place that described plant or its part can grow from this plant is removed to produce product.Typically, after plant grows up to, the required part gathered in the crops is removed from described plant, if be feasible in recirculation, and make product from the part gathered in the crops of described plant.During each enforcement method of the present invention, only can implement the step of a planting plants, allow the generation product step of multiplicity, and if the part gathered in the crops such as by repeatedly removing plant of the present invention needs to process these parts further to obtain product simultaneously.Also likely repeat to plant the step of plant of the present invention and store plant and maybe can gather in the crops part until carry out once the production of described product to the plant of accumulation or plant part.The step of side by side or continuously carrying out planting plants overlappingly even to a great extent and producing described product can be had in time.Usual described plant is grown up to by for some time before producing at product.

In one embodiment, the product produced by method of the present invention is plant prod, such as, but not limited to, food, feed, supplements, feed additive, fiber, makeup or medicine.Food is considered to the composition for nutrition or extra-nutrition.Animal-feed and animal-feed additive, in particular, be considered to food.In another embodiment, by production method of the present invention for the manufacture of agricultural prods such as, but be not limited to, plant milk extract, protein, amino acid, carbohydrate, fat, oil, polymkeric substance, VITAMIN etc.Plant prod may be made up of one or more agricultural prods to a great extent.

Can be unifacial leaf or dicotyledons according to the plant of different aspect of the present invention.Dicotyledons can be selected from such section, it includes but not limited to: composite family (Asteraceae), Cruciferae (Brassicaceae) (such as colea (Brassica napus)), Chenopodiaceae (Chenopodiaceae), Curcurbitaceae (Cucurbitaceae), pulse family (Leguminosae) (Caesalpiniaceae (Caesalpiniaceae), Aesalpiniaceae, Mimosaceae (Mimosaceae), Papilionaceae (Papilionaceae) or pulse family (Fabaceae)), Malvaceae (Malvaceae), the Rosaceae (Rosaceae) or Solanaceae.Such as, described plant can be selected from lettuce, Sunflower Receptacle, Arabidopis thaliana, cabbage, spinach, watermelon, pumpkin, wild cabbage, tomato, potato, capsicum, tobacco, cotton, rape (oilseed rape), gumbo, apple, rose, strawberry, clover, French beans, soybean, broad bean (field (fava) bean), pea, root of Szemao crotalaria, peanut, garbanzo, apricot, pear tree, peach, grape vine or Citrus species.In one embodiment, described plant is tobacco.In one embodiment, described plant is barley.In one embodiment, described plant is soybean.In one embodiment, described plant is cotton.In one embodiment, described plant is Semen Maydis.In one embodiment, described plant is paddy rice.In one embodiment, described plant is that rape comprises canola.In one embodiment, described plant is wheat.In one embodiment, described plant is sugarcane.In one embodiment, described plant is sugar beet.

What comprise equally is that biofuel and bioenergy crops such as rape (rape)/canola, Semen Lini, lupine and willow, willow, hybridization willow, switchgrass, awns belong to or gymnosperm, such as torch pine.What comprise equally is such crop, described crop is used for ensiling (corn), herbage or feed (grass, trifolium, sainfoin (sanfoin), clover), fiber (such as cotton, flax), building materials (such as pine tree, Oak Tree), paper pulp (such as willow), for raw material (feeder stocks) (the such as high erucic acid rape of chemical industry, Semen Lini) and pleasant (amenity) purposes (such as the turf of golf course), ornament (the such as Common Snapdragon of public and home garden, petunia, rose, Flos Pelargonii, Nicotiana species) and family expenses plant and cut-flower (African violet, Flower of Evans Begonia, chrysanthemum, Flos Pelargonii, Coleus (Coleus) bracketplant, any of several broadleaf plants (Dracaena) in thousand, rubber tree).

The present invention relates to trees in another embodiment, such as willow or eucalyptus.

Monocotyledons is passable, such as, is selected from following section: Palmae (Arecaceae), Amaryllidaceae (Amaryllidaceae) or Gramineae (Poaceae).Such as, described plant can be cereal crop, such as wheat, paddy rice, barley, corn, oat, Chinese sorghum, rye, onion, leek, millet, buckwheat, turf, Itanlian rye (Italian rye grass), switchgrass, awns, sugarcane or festuca species.

Preferably, described plant is crop plants.Crop plants refer to for human or animal consumption use or other non-food/feed applications commercial scale plantation any plant.The unrestricted example of crop plants comprises soybean, beet, sugar beet, Sunflower Receptacle, rape (comprises canola, witloof, Radix Dauci Sativae, cassava, clover, trefoil, coleseed, Semen Lini, cotton, tomato, potato, tobacco, willow, eucalyptus, pine tree, sugarcane and cereal such as paddy rice, corn, wheat, barley, millet, rye, triticale, Chinese sorghum, emmer, spelt (spelt), secale, einkorn, eragrosits abyssinica (teff), milo (milo) and oat.

Preferred plant is tobacco, corn, wheat, paddy rice, rape, Chinese sorghum, soybean, potato, tomato, barley, pea, French beans, cotton, broad bean, lettuce, cabbage or other Vegetable-type rapes (vegetable brassicas) or willow.In another embodiment, plant of the present invention and the plant that uses in the method for the invention are selected from the group be made up of the following: corn, paddy rice, wheat, soybean, cotton, rape (comprising canola), sugarcane, sugar beet and clover.

For the method for Plant Transformation, such as, by agrobacterium-mediated conversion or particle bombardment, and subsequently for regenerating and selecting the technology of plant through transforming to be widely known by the people in the art.Also it is within the scope of the invention that via the chloroplast transformation of biolistic (biobalistics).

According to various aspects of the invention, plant stress response is enhanced, strengthens or improves.This is understood to mean the enhancing compared to institute's discovery level in wild-type plant.In addition, as shown in Example, compared to the stress response of transgenic plant of core sequence of only expressing coding Fld, described level is also enhanced.Understanding can be measured this stress response to technician and described enhancing can be 2 to 10 times.There is coercing of two types: biotic is applied by other biological such as pathogenic agent, and abiotic stress results from excessive or not enough physics or chemical environment, such as arid, salinity, high or low temperature or Gao Guang.

The generation of chemical active ingredient such as ROS/RNS and removing are important for the physiological response in extensive biology and abiotic stress and plant.Reoxidizing after oxidative stress can bring out such as hyperoxia, light, arid, high salinity, cold, metal ion, pollutent, xenobiotic, toxin, anoxic by following multiple environment and biological factor, experimental implementation, pathogenic infection and plant organ aging.

Therefore, the invention particularly relates to for strengthening or strengthening the method for plant to the response of oxidative stress, such as, combine by extreme temperature, arid, UV-light, radiation, high salinity, cold, metal ion, pollutent, toxin or the pathogenic infection caused by bacterium, virus or fungi or its oxidative stress caused.

In another embodiment, method of the present invention and plant of the present invention relate to reinforcement to the patience of coercing being selected from the group be made up of the following: arid, less than 15 DEG C and low temperature above freezing, freezing temperature, salt stress, nutrient restriction, heavy metal stress, pathogenic infection and combination thereof.

In another aspect, the present invention relates to for reducing the method that in plant, ROS measures in the response of coercing, described method is included in plant expresses flavodoxin polypeptide and ferredoxin NADP (H) reductase enzyme polypeptide.According to this method, the construct that the Fld of guidance as described herein and FNR expresses can be used.Alternatively, the plant of expression Fld can be made with the plant hybridization of expressing FNR thus obtain the coexpression of two kinds of genes.

In another aspect of the present invention, require the right of such method, described method is used under stress conditions, such as can gather in the crops part, flower or seed compared to control plant increase plant or plant part chlorophyll and/or carotenoid levels.

According to embodiment of the present invention, the relative expression levels of Fld and FNR can with the effect change directly depending on Fld dosage.In preferred embodiments, the expression level of Fld is at least identical with the expression level of ferredoxin.

Embodiment

The present invention will be described in the following non-limiting examples.

Method

Vector construction

Build for express FNR Ti carrier and in tobacco coexpression Fld and FNR

In cyanobacteria, FNR is albumen in film, it is made up of three structural domains: FAD binding domains, NADP (H) binding domains and with the interactional integrated structure territory of phycobilisome (Fillat etc., 1993), and the first two structural domain can be separated with described inner domain by proteolysis or mutagenesis, produce the albumen of two solvable structural domains, described albumen retains comprehensive NADPH-ferredoxin (Fld) active (Mart í nez-J ú lvez etc., 1996).This engineering should ensure that cyanobacteria FNR will keep solvable and only needed for displaying activity in the chloroplast stroma of transgenic plant.Therefore we process the FNR gene from fish raw meat cyanobacteria PCC7119.Make the 3rd structural domain disappearance and the mosaic gene of gained is introduced tobacco.After making FNR plant and the compatriot expressing Fld hybridize, two homozygote plant is selected and is closed the larger patience of Fld plant display to methyl viologen (MV) toxicity compared to simple.

Whole gene (the Fillat etc. in plasmid pTrc99a are cloned into by pcr amplification, 1990), use respectively with primer (primer 1) 5 '-CCGAGCTCACACCATGACTCAAGCGAA-3 ' (SEQ ID NO 11) and (primer 2) 5 '-ACGTCGACCAACTTAGTATGTTTCTAC-3 ' (SEQ ID NO 12) of position 1 to 19 and 906 to 925 complementation, obtain the DNA fragmentation of encoding from the region of the FNR (not there is phycobilisome binding domains) of fish raw meat cyanobacteria PCC7119.For convenience of operation further, SacI recognition site (GAGCTC) is incorporated into primer 15 ' holds and SalI site (GTCGAC) is incorporated into 3 ' end of primer 2.PCR condition is: 94 DEG C of 60s, 54 DEG C of 60s and 72 DEG C 90s, and 30 circulations are comprising 10mM Tris-HCl pH 8.4,5mM KCl, 1.5mM MgCl ₂, 0.2mM often plants in the medium of dNTP and 2.5 unit Taq archaeal dna polymerases and uses 1ng template DNA and each primer of 50pmol.After completing 30 circulations, make reaction at 72 DEG C of incubation 10min.The PCR fragment with prediction length (940bp) of purifying is digested with SacI and SalI.The pUC9 described fragment being cloned into whole pea FNR precursor of encoding between BamHI and SalI restriction site derives recombinant plasmid (Ceccarelli etc., 1991) compatible sites, and the DNA fragmentation in the ripe region of the pea FNR that encodes is by removing from described plasmid with SacI and SalI digestion.This produces the frame endomixis being derived from the chloroplast transit peptides of pea FNR and the ripe region of fish raw meat cyanobacteria FNR.

Two chains are determined the sequence of mosaic gene, and by using BamHI and SalI digestion it to be excised from corresponding plasmid.Then between the CaMV 35S promoter described 1120-bp fragment being cloned into pDH51 (Pietrzcak etc., 1986) and polyadenylation region.Whole box is separated as EcoRI fragment and is inserted into the EcoRI site of binary vector pCAMBIA 2200 (Hajdukiewiez etc., 1994) further.Described construct is made to enter Agrobacterium tumefaciens (Agrobacterium tumefaciens) bacterial strain GV3101 pMP 90 (Ausubel etc., 1987) finally by electroporation.

Build Ti carrier for expressing FNR and Fld, and in barley coexpression Fld and FNR

Develop two separate carrier for the experimental procedure with FNR and Fld cotransformation barley plants according to (2009) such as Harwood.All molecular biology recombinant DNA technologies related to are known to technician and obtain full-time instruction in the literature.Hold the sequence of the mosaic gene of the frame endomixis of the coding region of two structural domains (SEQ ID NO.4) to produce binary vector (the Harwood et al being suitable for being cloned into pBRACT series by the C comprising chloroplast transit peptides and the fish raw meat cyanobacteria PCC7119 FNR deriving from pea FNR described before pcr amplification, 2009) product in, the binary vector of described pBRACT series comprises hpt gene, and described hpt gene gives hygromycin resistance under the control of 35S promoter being positioned at left border (LB).The gene of described chimerical clone is under the control of maize ubiquitin promoter being positioned at right side boundary (RB).Make to comprise Fld coding region (SEQ ID NO.2, Tognetti etc., 2006 of chloroplast transit peptides and the fish raw meat cyanobacteria PCC7119 deriving from pea FNR; The chimeric constructs of frame endomixis PCT/GB2002/004612), experiences the experimental procedure identical with the above.

The binary vector of the goal gene be included under the required control regulating sequence of gained can be directly used in Plant Transformation step, such as agrobacterium-mediated plant tissue transformation or particle bombardment technology.

Build the binary vector being used for coexpression Fld and FNR in plant

Based on MultiSite Gateway cloning system (Invitrogen, http://www.invitrogen.com) (Karimi etc. 2007; Dafny-Yelin and Tzfira, 2007), develop so single construct, described single construct can instruct FNR and Fld polypeptide transforming the coexpression had in the plant of such construct.Figure 11 describes design and builds the multi-step process of above-mentioned complex carries.Described process is carried out in operation instruction, experimental program and guidance that deferring to is provided by manufacturers.All molecular biology recombinant DNA technologies related to are known to technician and obtain full-time instruction in the literature.

Hold the sequence of the mosaic gene of the frame endomixis of the coding region of two structural domains (SEQ ID NO.4) to produce the product be suitable for use as with the substrate in the Gateway BP recombining reaction of suitable donor vehicle by the C comprising chloroplast transit peptides and the fish raw meat cyanobacteria PCC7119 FNR deriving from pea FNR described before pcr amplification.The primer of design two gene specifics, forward with reverse, thus respectively attB1 and attB4 sequence being attached to its 5 ' end, described attB1 and attB4 sequence is with required for the specific BP recombining reaction in attP1 and the attP4 site in pDONR221 P1-P4 donor vehicle.Locus specificity BP recombining reaction between attB1-FNR-attB4 PCR primer and pDONR221 P1-P4 carrier produces pENTR221 L1-L4-FNR and enters clone, and wherein the both sides of FNR construct are attL1 and the attL4 locus specificity sequences for LR restructuring.Make to comprise Fld coding region (SEQ ID NO.2, Tognetti etc., 2006 of chloroplast transit peptides and the fish raw meat cyanobacteria PCC7119 deriving from pea FNR; The chimeric constructs of frame endomixis PCT/GB2002/004612), experiences the experimental procedure similar to the above, except primer to be recombinated in conjunction with attB2 and attB3 attB1 and the attB4 site of specific sequence instead of construct before.BP recombining reaction between the attB2-Fld-attB3 PCR primer of gained and pDONR221 P2-P3 donor vehicle produces pENTR221 L2-L3-Fld and enters clone, and wherein the both sides of Fld construct are attL2 and attL3 LR restructuring specific position.

PENTR221 L1-L4-FNR and pENTR221 L2-L3-Fld is entered clone and be used as the substrate with the MultiSite Gateway LR recombining reaction of any multiple specially designed pDEST-BRACT object carrier (pBRACT).PDEST-BRACT carrier is MultiSite Gateway object carrier, it is transformed thus is comprised two Gateway boxes, and described two Gateway boxes are used for there are two different constructs of compatible attL sequence at pre-determined direction by single LR Site-specific recombinase reaction independent cloning flank.They are binary T-DNA vectors, it is except left side and right side T-DNA border sequence (LB and RB, respectively) outward, also comprise complete plant selectable marker expression cassette and plant regulation domain (promotor, terminator, enhanser), it is positioned at the both sides of each Gateway box thus instructs the expression being cloned sequence.The promotor of the multiple pDEST-BRACT object carrier of exploitation is different with the identity of the attL sequence that terminator and/or its comprise.They can be customized to in unifacial leaf or dicotyledons under the control of composing type or inducible promoter transgenosis described in optimal expression.

The cloning by expression of gained be included in required regulate sequence to control under the binary vector of goal gene, it can be directly used in Plant Transformation step, such as agrobacterium-mediated plant tissue transformation or particle bombardment technology.

Fld and FNR is expressed in tobacco

Plant Transformation

Use routine techniques (Gallois and Marinho, 1995) to implement tobacco (Nicotiana tabacum cv Petit Havana) leaf dish to transform and the offspring analyzing kalamycin resistance transformant further.The primary transformant self-pollination of the high-level cyanobacteria FNR that expression is assessed as SDS-PAGE and immunoblotting, and use offspring of isozygotying to carry out all experiments subsequently.

Express the generation of the transgenic plant of Fld and FNR from fish raw meat cyanobacteria simultaneously

The preparation of double expression(DE) plant is carried out by cross-pollination.Be used in transgenic plant that produce in this project, that express the FNR (pFNR) from fish raw meat cyanobacteria, with the stable homozygous line (pFld of high level expression fish raw meat cyanobacteria Fld in chloroplast(id), Tognetti etc., 2006) as parent.Make the elementary pair of heterozygosis transformant self-pollination of expression pFNR and pFld and select two homozygote plant by SDS-PAGE and immunoblotting.

Stress treatment

Make the seed of contrast and transgenic plant 100 μ g ml when being supplemented with 3% (w/v) sucrose and in transformant ^-1murashige-Skoog (MS) agar of kantlex germinates.At 4 weeks 25 DEG C and 100 μm of ol quanta m ^-2s ^-1after (16h illumination/8h is dark), seedling is placed on soil.Punch out from the leaf launched completely that the two monthly age tobacco plants of growth on soil are tender the leaf dish of 13mm diameter.Weigh to leaf dish and make its top side swim in comprising on the 1ml sterile distilled water of the MV of amount shown in 24 orifice plates up, individually, and in 25 DEG C in the dark incubation 12h be diffused in leaf to allow MV.Then with 700 μm of ol quanta m ^-2s ^-1white light source irradiate hole.Remain in water under impinging upon similarity condition.Use Horiba Mode B-173 mhometer, during MV coerces, the electrolyte leakage of leaf dish is measured as the specific conductivity of medium.

The seedling that grown 3 or 4 weeks in soil is transferred in the hydroponic system comprising Hoagland solution (Hoagland and Arnon, 1950).After 3 days, supplement to Hoagland solution with 100 μMs of MV.

Analytical procedure

Pigment detection

Use the Chlorophylls and Carotenoids content in standard method (Lichtenthaler, 1987) mensuration leaf and plastid.

Detect lipid peroxide

Use FOX to measure to quantize the existence (DeLong, etc., 2002) of lipid peroxide (LOOHs) in plant tissue extract.300 μ L are used to comprise 80: 20 (v/v) ethanol of 0.01% Butylated Hydroxytoluene: water extraction leaf texture (4cm ²).Lipid is assigned to organic phase, and vortex oscillation is also centrifugal with 3,000g.Plant milk extract described in 50 μ l and 50 μ l 10mM tris-phosphniline (TPP, LOOH reductive agent) are incorporated in methyl alcohol and 500U beef liver catalase (Sigma).Stir described mixture and incubation 30min is reduced by TPP (+TPP) completely to make-the OOH of any existence.Process the sample (-TPP) not adding TPP in the same manner, except replacing except TPP aliquots containig with methyl alcohol.After 30min TPP incubation, by 900 μ l by 90% methyl alcohol (v/v), 25mM H ₂sO ₄, 4mM Yoshinox BHT (BHT), 25 μMs of ferrous ammonium sulphate hexahydrates and 100 μMs of xylenol orange composition FOX reagent add each sample to, and after adding 10min, in Ultrospec 1100 spectrophotometer (Amersham, Biosciences), record the absorbancy at 560nm place.Existence containing the absorbance difference display LOOH with the sample room not containing TPP; Then use and cross over 0-20 μM of H ₂o ₂-OOH value is expressed as micromole H by the typical curve of scope ₂o ₂equivalent.

Enzyme assay

For the enzyme of the diaphorase activity that NADPH relies on is shown in qualification, on 12% polyacrylamide gel, be separated the leaf extract being equivalent to 15 μ g soluble proteins by Native PAGE.After electrophoresis, by 50mM Tris-HCl, pH 8.5,0.3mM NADP ⁺, 3mM Glc-6-P, 1 unit nl ^-1glc-6-P desaturase and 1mg ml ^-1in nitroblue tetrazolium(NBT), incubation is given described gel-colored until there is the first of purple band.

The enzymic activity of method determination ascorbate peroxidase enzyme (APX) of Mittler and Zilinskas (1993) is used in native gel.

Result

Solvable fish raw meat cyanobacteria FNR is expressed in transgene tobacco chloroplast(id)

In order to express solvable cyanobacteria FNR in Nicotiana tabacum plastid, prepare such mosaic gene, wherein C holds fish raw meat cyanobacteria FNR coding region (Fillat etc., 1990) of two structural domains at aminoterminal by the DNA sequence dna (details square method part) of frame endomixis to the chloroplast transit peptides of coding pea FNR.Described construct is cloned in the edaphic bacillus binary vector under composing type CaMV 35S gene promoter controls, and is delivered in tobacco cell by the conversion of agrobacterium-mediated leaf dish.Kalamycin resistance plant is regained from tissue culture and is accumulated by immunoblotting assessment FNR.By SDS-PAGE separation and Extraction from the primary transformant (pFNR) sampled or the albumen extracted from wild-type tobacco sample, and with coomassie brilliant blue staining, or on trace to nitrocellulose filter and use standard technique with for fish raw meat cyanobacteria FNR antiserum(antisera) detect (Fig. 2).

Obtaining the activity band that the ripe size of different levels can be detected in the leaf extract of some transformant, hint plastid inputs and processes the flavoprotein of expressing.While FNR being detected in the matrix at transgenic plant chloroplast(id), in thylakoid membrane fraction, there is no immunoreactivity (Fig. 3 A).It is activated (Fig. 3 B) that the diaphorase activity of chloroplast stroma fraction is presented at enzyme described in rotaring gene tobacco plant.

The plant versus wild type compatriot in phenotype expressing cyanobacteria FNR in chloroplast(id) looks like normally, and shows the patience (data are not shown) to the wild-type levels of MV toxicity.

Fish raw meat cyanobacteria FNR and Fld is expressed in transgene tobacco chloroplast(id)

For obtaining double expression(DE) plant, between the homozygote plant of expressing FNR or FLD, carry out cross-pollination.The offspring of gained only includes two heterozygosis sample as expected.Make its self-pollination and select two (2x) plant (Fig. 4) of isozygotying by western blot method.

To the patience of methyl viologen

Carry out testing to assess FNR/Fld and express leaf dish to the patience of MV, as described in method part.Be visually observed leaf texture's bleaching at contrast Ye Panzhong, reflect the chlorophyll degradation (Fig. 5 A) of increase.By measuring the membrane damage that electrolyte leakage assessment exposes owing to MV.Electric conductivity value is revised the Ion leakage occurred under the same conditions in water and is expressed as the per-cent of total ion content (maximum value the ending of MV process obtains after autoclaving leaf dish).Chlorophyll content is expressed as the mark of the Chlorophyll of the leaf dish of incubation under the same terms not having MV.Film rotten (deterioration) and pigment integrity are compared and are all obtained better protecting significantly (Fig. 5 B, C) simple conjunction in Fld expression compatriot in the two FNR/Fld of isozygotying plant.

For assessing whole strain plant to the patience of MV, such as in the hydroponic system described in method, measuring them.Compared to only expressing Fld, expressing while FNR and Fld and the more protection (Fig. 6) to the damage that MV brings out is provided.

For assessment ROS increases, measured by FOX and measure lipid peroxidation (Delong etc., 2002).As the leaf dish with 10 μMs of MV process wild-types and rotaring gene tobacco plant described in method.The level of lipid hydroperoxide (LOOH) is with a μM H ₂o ₂cm ^-2represent, and two isozygoty hybridization X416 in than isozygoty lower significantly in parent pFld.Both have more patience (Fig. 7 A) than wild-type plant.Some albumen is also the preferred target of ROS.Chloroplast(id) ascorbate peroxidase enzyme (APX) is wherein the most responsive.Wild-type plant is exposed to the decline that 20 μMs of MV cause this enzymic activity 70-80% after only 90min incubation.The expression providing unit of Fld divides protection (residual activity of 40-50%).The maintenance (Fig. 7 B) causing APX activity almost quantitative is there is in FNR while Fld expresses in plant.

The expression of Fld and FNR in barley and coexpression

Plant Transformation.Produce the transgenic Barley plant of Fld and FNR simultaneously expressed from fish raw meat cyanobacteria.

Use the pBract214 vector barley comprising Fld and FNR gene respectively, as mentioned above.Described carrier is transformed into independently in Agrobacterium tumefaciens and with the mixture of two kinds of edaphic bacillus strains and transforms spring barley mutation Golden Promise.Barley transformation is carried out in infection based on the prematurity plumule with Agrobacterium tumefaciens, selects genetically modified organism subsequently on the substratum comprising antibiotic hygromycin.The method causes the generation (Harwoodd etc., 2009) of the separate transgenic strain of fecund and the offspring of the transformant of hygromycin resistance is further analyzed.To the elementary heterozygosis transformant of cyanobacteria FNR and Fld be expressed, as with SDS-PAGE and immunoblotting assessed, for further experiment.In the amendment to experimental program as herein described, only carry out the infection of plumule with to be equipped with in the Agrobacterium tumefaciens strain of a cyanobacteria gene construct each to obtain the independently Heterozygous transgenic strain of expression FNR or Fld construct.

Stress treatment in barley

Render transgenic and contrast barley plants grow under controlled envrionment conditions, described controlled envrionment conditions refer to daytime 15 DEG C and night the temperature of 12 DEG C, the humidity of 80%, 16h photoperiod provided by metal halide lamp (HQI) and in maturation plant canopy level with 500 μm of ol quanta m ^-2s ^-1intensity supplement with tungsten lamp.Levington M3 mixed fertilizer/Perlite/Grit that soil mixture used is mixed by the ratio with 2: 2: 1 forms.From growth soil 6 weeks age barley plants leaf cut 10-15mm grow leaf bar.Then make leaf bar in the distilled water of the MV and 0.05% Tween-20 that comprise amount shown 20 DEG C in the dark incubation within 30 minutes, be diffused in tissue to make MV.Then paraxial for described leaf bar side direction Shangdi to be placed in plastic pallet and at 450 μm of ol quanta m ^-2s ^-1time cycle under light source shown in incubation.Contrast is remained on comprise in the distilled water of 0.05%Tween-20.Then as described in 5.1, assess the content of Chlorophylls and Carotenoids.

Result

Make the independent heterozygosis barley plants of expression Fld with FNR obtained according to methods described herein experience oxidative stress conditions to assess their relative resistance compared with its wild type counterparts.Figure 12 display when for FNR and Fld gene be the transgenic plant of heterozygosis and the leaf bar of wild-type individuality be exposed to oxidation reduction cycle weedicide methyl viologen time the typical consequence that obtains and then estimate the content of photosynthetic pigments chlorophylls and carotenoid as described in method.Pigment degradation is the mark that photosynthetic organs goes bad.Result shows, and two heterozygosis FNR/Fld transgenic plant successfully subjected to oxidation challenge, and the level of its total Chlorophylls and Carotenoids preserved is respectively than the corresponding object height 2 times of wild-type (and independent FNR) and 4 times.Tolerant levels in the middle of the display of Fld express transgenic barley plants.Consider the fact of the dose-dependently of the protected effect given by transgenosis, the heterozygote plant of two kinds of transgenosis FNR and Fld shows that the fact of high tolerant levels is significant.

Conclusion

From the patience to MV toxicity expressing the enhancing of the plant giving relative only expression Fld while Fld with FNR of identical cyanobacteria species is in plant.For the sake of simplicity, pn plant represents elementary FNR tobacco Transformant, and Xn plant is pn plant and the hybridization from the pfld5-8 of (2006) such as Tognetti.Xnn or Xnnn is the two autophilous segregant of heterozygote plant (segregant) of X4.

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Sequence table

Nucleotide sequence as herein described and corresponding peptide list under.

Seq 1: without the Fld nucleotide sequence for single fusion constructs of target sequence

ATGTCAAAGAAAATTGGTTTATTCTACGGTACTCAAACTGGTAAAACTGAATCAGT

AGCAGAAATCATTCGAGACGAGTTTGGTAATGATGTGGTGACATTACACGATGTTT

CCCAGGCAGAAGTAACTGACTTGAATGATTATCAATATTTGATTATTGGCTGTCCT

ACTTGGAATATTGGCGAACTGCAAAGCGATTGGGAAGGACTCTATTCAGAACTGG

ATGATGTAGATTTTAATGGTAAATTGGTTGCCTACTTTGGGACTGGTGACCAAATA

GGTTACGCAGATAATTTTCAGGATGCGATCGGTATTTTGGAAGAAAAAATTTCTCA

ACGTGGTGGTAAAACTGTCGGCTATTGGTCAACTGATGGATATGATTTTAATGATT

CCAAGGCACTAAGAAATGGCAAGTTTGTAGGACTAGCTCTTGATGAAGATAATCAA

TCTGACTTAACAGACGATCGCATCAAAAGTTGGGTTGCTCAATTAAAGTCTGAATT

TGGTTTGTAA

Seq 2: with the Fld nucleotide sequence for single fusion constructs of target sequence

ATGGCTGCTGCAGTAACAGCCGCAGTCTCCTTGCCATACTCCAACTCCACTTCCC

TTCCGATCAGAACATCTATTGTTGCACCAGAGAGACTTGTCTTCAAAAAGGTTTCA

TTGAACAATGTTTCTATAAGTGGAAGGGTAGGCACCATCAGAGCTCTCATAATGTC

AAAGAAAATTGGTTTATTCTACGGTACTCAAACTGGTAAAACTGAATCAGTAGCAG

AAATCATTCGAGACGAGTTTGGTAATGATGTGGTGACATTACACGATGTTTCCCAG

GCAGAAGTAACTGACTTGAATGATTATCAATATTTGATTATTGGCTGTCCTACTTGG

AATATTGGCGAACTGCAAAGCGATTGGGAAGGACTCTATTCAGAACTGGATGATG

TAGATTTTAATGGTAAATTGGTTGCCTACTTTGGGACTGGTGACCAAATAGGTTAC

GCAGATAATTTTCAGGATGCGATCGGTATTTTGGAAGAAAAAATTTCTCAACGTGG

TGGTAAAACTGTCGGCTATTGGTCAACTGATGGATATGATTTTAATGATTCCAAGG

CACTAAGAAATGGCAAGTTTGT?AGGACTAGCTCTTGATGAAGATAATCAATCTGAC

TTAACAGACGATCGCATCAAAAGTTGGGTTGCTCAATTAAAGTCTGAATTTGGTTT

GTAA

Seq 3 belongs to PCC7119 nucleotide sequence (sequences of coding two structural domains) without the FNR fish raw meat cyanobacteria for single fusion constructs of target sequence

ATGACTCAAGCGAAAGCCAAACACGCTGATGTTCCTGTTAATCTTTACCGTCCCAA

TGCTCCATTTATTGGTAAGGTAATCTCTAATGAACCACTGGTAAAAGAAGGCGGGA

TAGGTATTGT?TCAGCACATTAAATTTGATCTAACTGGTGGTAACTTAAAGTACATCG

AAGGTCAAAGTATTGGTATCATTCCACCAGGAGTGGACAAGAACGGCAAGCCGGA

AAAATTGAGACTCTACTCCATTGCCTCGACCCGTCACGGCGATGATGTGGATGAT

AAAACCATCTCACTGTGCGTCCGTCAATTAGAGTACAAACATGCAGAAAGCGGCG

AAACAGTTTACGGTGTTTGTTCTACTTACTTGACTCACATTGAACCAGGTTGAGAA

GTGAAAATCACTGGGCCTGTGGGTAAAGAAATGCTGTTACCCGATGATCCTGAAG

CTAATGTCATCATGTTGGCAACAGGTACTGGTATTGCGCCTATGCGGACTTACCT

GTGGCGGATGTTCAAGGATGCAGAAAGAGCTGCTGACCCAGAATATCAATTCAAA

GGATTCTCTTGGTTAGTCTTTGGTGTTCCTACAACTCCTAACATTCTTTATAAAGAA

GAAGTGGAAGAAATCCAACAAAAATATCCCGATAACTTCCGCCTAACTTACGCTAT

CAGCCGGGAGCAAAAGAATCCCCAAGGTGGCAGAGTGTACATCCAAGACCGTGT

GGCAGAACACGCTGATGAACTGTGGCAATTAATCAAGAATGAAAAAACCCACACC

TACATCTGTGGTTTGCGCGGTATGGAAGAGGGCATTGATGCTGCTTTAAGTGCTG

CGGCTGCGAAAGAAGGTGTTACCTGGAGTGATTACCAAAAAGACCTCAAGAAAGC

TGGTCGCTGGCACGTAGAAACATACTAA

Seq 4 is with the FNR nucleotide sequence for single fusion constructs (comprising the FNR construct of the sequence of coding two structural domains) of target sequence

ATGGCTGCTGCAGTAACAGCCGCAGTCTCCTTGCCATACTCCAACTCCACTTCCC

TTCCGATCAGAACATCTATTGTTGCACCAGAGAGACTTGTCTTCAAAAAGGTTTCA

TTGAACAATGTTTCTATAAGTGGAAGGGTAGGCACCATCAGAGCTCACACCATGA

CTCAAGCGAAAGCCAAACACGCTGATGTTCCTGTTAATCTTTACCGTCCCAATGCT

CCATTTATTGGTAAGGTAATCTCTAATGAACCACTGGTAAAAGAAGGCGGGATAG

GTATTGTTCAGCACATTAAATTTGATCTAACTGGTGGTAACTTAAAGTACATCGAAG

GTCAAAGTATTGGTATCATTCCACCAGGAGTGGACAAGAACGGCAAGCCGGAAAA

ATTGAGACTCTACTCCATTGCCTCGACCCGTCACGGCGATGATGTGGATGATAAA

ACCATCTCACTGTGCGTCCGTCAATTAGAGTACAAACATCCAGAAAGCGGCGAAA

CAGTTTACGGTGTTTGTTCTACTTACTTGACTCACATTGAACCAGGTTCAGAAGTG

AAAATCACTGGGCCTGTGGGTAAAGAAATGCTGTTACCCGATGATCCTGAAGCTA

ATGTCATCATGTTGGCAACAGGTACTGGTATTGCGCCTATGCGGACTTACCTGTG

GCGGATGTTCAAGGATGCAGAAAGAGCTGCTGACCCAGAATATCAATTCAAAGGA

TTCTCTTGGTTAGTCTTTGGTGTTCCTACAACTCCTAACATTCTTTATAAAGAAGAA

CTGGAAGAAATCCAACAAAAATATCCCGATAACTTCCGCCTAACTTACGCTATCAG

CCGGGAGCAAAAGAATCCCCAAGGTGGCAGAGTGTACATCCAAGACCGTGTGGC

AGAACACGCTGATGAACTGTGGCAATTAATCAAGAATGAAAAAACCCACACCTACA

TCTGTGGTTTGCGCGGTATGGAAGAGGGCATTGATGCTGCTTTAAGTGCTGCGGC

TGCGAAAGAAGGTGTTACCTGGAGTGATTACCAAAAAGACCTCAAGAAAGCTGGT

CGCTGGCACGTAGAAACATACTAA

The nucleotide sequence (comprising 3 structural domains) that Seq 5:FNR is complete

ATGTCTAATCAAGGTGCTTTTGATGGTGCTGCCAACGTAGAATCAGGTAGCCGCG

TCTTCGTTTACGAAGTGGTGGGTATGCGTCAGAACGAAGAAACTGATCAAACGAA

CTACCCAATTCGTAAAAGTGGCAGTGTGTTCATTAGAGTGCCTTACAACCGCATGA

ATCAAGAAATGCAGCGTATCACTCGACTAGGCGGCAAGATTGTTACGATTCAAAC

AGTAAGCGCACTACAACAACTCAATGGTAGAACTACCATTGCAACAGTAACAGATG

CGTCTAGTGAGATTGCTAAGTCTGAGGGGAATGGTAAAGCCACACCTGTAAAAAC

TGATAGTGGAGCTAAAGCGTTCGCTAAACCACCAGCTGAAGAACAGCTTAAGAAA

AAAGACAACAAAGGCAACACCATGACTCAAGCGAAAGCCAAACACGCTGATGTTC

CTGTTAATCTTTACCGTCCCAATGCTCCATTTATTGGTAAGGTAATCTCTAATGAAC

CACTGGTAAAAGAAGGCGGGATAGGTATTGTTCAGCACATTAAATTTGATCTAACT

GGTGGTAACTTAAAGTACATCGAAGGTCAAAGTATTGGTATCATTCCACCAGGAGT

GGACAAGAACGGCAAGCCGGAAAAATTGAGACTCTACTCCATTGCCTCGACCCGT

CACGGCGATGATGTGGATGATAAAACCATCTCACTGTGCGTCCGTCAATTAGAGT

ACAAACATCCAGAAAGCGGCGAAACAGTTTACGGTGTTTGTTCTACTTACTTGACT

CACATTGAACCAGGTTCAGAAGTGAAAATCACTGGGCCTGTGGGTAAAGAAATGC

TGTTACCCGATGATCCTGAAGCTAATGTCATCATGTTGGCAACAGGTACTGGTATT

GCGCCTATGCGGACTTACCTGTGGCGGATGTTCAAGGATGCAGAAAGAGCTGCT

GACCCAGAATATCAATTCAAAGGATTCTCTTGGTTAGTCTTTGGTGTTCCTACAAC

TCCTAACATTCTTTATAAAGAAGAACTGGAAGAAATCCAACAAAAATATCCCGATAA

CTTCCGCCTAACTTACGCTATCAGCCGGGAGCAAAAGAATCCCCAAGGTGGCAGA

GTGTACATCCAAGACCGTGTGGCAGAACACGCTGATGAACTGTGGCAATTAATCA

AGAATGAAAAAACCCACACCTACATCTGTGGTTTGCGCGGTATGGAAGAGGGCAT

TGATGCTGCTTTAAGTGCTGCGGCTGCGAAAGAAGGTGTTACCTGGAGTGATTAC

CAAAAAGACCTCAAGAAAGCTGGTCGCTGGCACGTAGAAACATACTAA

SEQ 6:Fld aminoacid sequence

MSKKIGLFYGTQTGKTESVAEIIRDEFGNDVVTLHDVSQAEVTDLNDYQYLIIGCPTWN

IGELQSDWEGLYSELDDVDFNGKLVAYFGTGDQIGYADNFQDAIGILEEKISQRGGKT

VGYWSTDGYDFNDSKALRNGKFVGLALDEDNQSDLTDDRIKSWVAQLKSEFGL

SEQ 7:: with the Fld aminoacid sequence of target sequence

MAAAVTAAVSLPYSNSTSLPIRTSIVAPERLVFKKVSLNNVSISGRVGTIRALIMSKKIGL

FYGTQTGKTESVAEIIRDEFGNDVVTLHDVSQAEVTDLNDYQYLIIGCPTWNIGELQSD

WEGLYSELDDVDFNGKLVAYFGTGDQIGYADNFQDAIGILEEKISQRGGKTVGYWST

DGYDFNDSKALRNGKFVGLALDEDNQSDLTDDRIKSWVAQLKSEFGL

Seq 8: the FNR fish raw meat cyanobacteria without target sequence belongs to PCC7119 aminoacid sequence (2 structural domains)

MTQAKAKHADVPVNLYRPNAPFIGKVISNEPLVKEGGIGIVQHIKFDLTGGNLKYIEGQ

SIGIIPPGVDKNGKPEKLRLYSIASTRHGDDVDDKTISLCVRQLEYKHPESGETVYGVC

STYLTHIEPGSEVKITGPVGKEMLLPDDPEANVIMLATGTGIAPMRTYLWRMFKDAER

AADPEYQFKGFSWLVFGVPTTPNILYKEELEEIQQKYPDNFRLTYAISREQKNPQGGR

VYIQDRVAEHADELWQLIKNEKTHTYICGLRGMEEGIDAALSAAAAKEGVTWSDYQKD

LKKAGRWHVETY

Seq 9: the FNR fish raw meat cyanobacteria with target sequence belongs to PCC7119 aminoacid sequence (2 structural domains)

MAAAVTAAVSLPYSNSTSLPIRTSIVAPERLVFKKVSLNNVSISGRVGTIRAHTMTQAK

AKHADVPVNLYRPNAPFIGKVISNEPLVKEGGIGIVQHIKFDLTGGNLKYIEGQSIGIIPP

GVDKNGKPEKLRLYSIASTRHGDDVDDKTISLCVRQLEYKHPESGETVYGVCSTYLTH

IEPGSEVKITGPVGKEMLLPDDPEANVIMLATGTGIAPMRTYLWRMFKDAERAADPEY

QFKGFSWLVFGVPTTPNILYKEELEEIQQKYPDNFRLTYAISREQKNPQGGRVYIQDR

VAEHADELWQLIKNEKTHTYICGLRGMEEGIDAALSAAAAKEGVTWSDYQKDLKKAG

RWHVETY

Seq 10: the aminoacid sequence (3 domain sequence) that the FNR without target sequence is complete

MSNQGAFDGAANVESGSRVFVYEVVGMRQNEETDQTNYPIRKSGSVFIRVPYNRMN

QEMQRITRLGGKIVTIQTVSALQQLNGRTTIATVTDASSEIAKSEGNGKATPVKTDSGA

KAFAKPPAEEQLKKKDNKGNTMTQAKAKHADVPVNLYRPNAPFIGKVISNEPLVKEG

G?IGIVQHIKFDLTGGNLKYIEGQSIGIIPPGVDKNGKPEKLRLYSIASTRHGDDVDDKTIS

LCVRQLEYKHPESGETVYGVCSTYLTHIEPGSEVKITGPVGKEMLLPDDPEANVIMLA

TGTGIAPMRTYLWRMFKDAERAADPEYQFKGFSWLVFGVPTTPNILYKEELEEIQQKY

PDNFRLTYAISREQKNPQGGRVYIQDRVAEHADELWQLIKNEKTHTYICGLRGMEEGI

DAALSAAAAKEGVTWSDYQKDLKKAGRWHVETY

Claims

1. one kind for generation of the unifacial leaf of stress tolerance or the method for dicotyledons oxidative stress to reinforcement, described method is included in plant the nucleotide sequence of the nucleotide sequence of the flavodoxin polypeptide of expressing encoding bacterial and ferredoxin NADP (H) the reductase enzyme polypeptide of coding cyanobacteria, the nucleotide sequence of ferredoxin NADP (H) the reductase enzyme polypeptide of described coding cyanobacteria comprises the sequence that coding C holds two domain region, but does not comprise the sequence of coding phycobilisome binding domains.

2. method according to claim 1, described method is included in express nucleic acid construct in described plant, and wherein said nucleic acid construct comprises the nucleotide sequence of coding flavodoxin polypeptide and the nucleotide sequence of encoding ferredoxin NADP (H) reductase enzyme polypeptide.

3. method according to claim 2, wherein said construct instructs the coexpression of flavodoxin and ferredoxin NADP (H) reductase enzyme polypeptide.

4. method according to claim 1, described method comprises

A) express nucleic acid construct in plant, described construct comprises the sequence of coding flavodoxin polypeptide,

B) express nucleic acid construct in the second plant, described construct comprises the nucleotide sequence of coding FNR polypeptide,

C) the first and second plant hybridizations are made, and

D) the stable homozygote plant of expressing FNR and Fld is produced.

5. method according to claim 1, described method comprises

A) express nucleic acid construct in plant, described construct comprises the sequence of coding flavodoxin polypeptide or FNR polypeptide,

B) described plant is transformed, to produce the stable homozygote plant of expressing FNR and Fld with the nucleic acid construct of the sequence of the sequence or coding FNR polypeptide that comprise coding flavodoxin polypeptide respectively.

6. method according to claim 1, the nucleotide sequence of the flavodoxin polypeptide of wherein encoding a) derives from cyanobacteria and described flavodoxin polypeptide is cyanobacteria flavodoxin or b) derives from heterotrophic organism.

7. method according to claim 1, the nucleotide sequence of flavodoxin polypeptide of wherein encoding is selected from nucleotide sequence as shown in Table 1.

8. method according to claim 1, wherein the nucleotide sequence of encoding ferredoxin NADP (H) reductase enzyme polypeptide is selected from nucleotide sequence as shown in Table 2.

9. the method any one of claim 6 to 8, wherein said cyanobacteria is selected from: Crocosphaera, blue Pseudomonas (Cyanobium), blue silk Pseudomonas (Cyanothece), micro-capsule cyanobacteria belongs to (Microcystis), Synechococcus belongs to (Synechococcus), collection born of the same parents cyanobacteria belongs to (Synechocystis), Thermosynechococcus, microtriche cyanobacteria belongs to (Microchaetaceae), Nostocaceae, sheath silk cyanobacteria belongs to (Lyngbya), spiral cyanobacteria belongs to (Spirulina) or restraints darkish blue bacterium genus (Trichodesmium).

10. the method any one of claim 6 to 8, wherein said cyanobacteria is selected from: Fremyella, single discrimination cyanobacteria belongs to (Tolypothrix), fish raw meat cyanobacteria belongs to (Anabaena), necklace cyanobacteria belongs to (Anabaenopsis), blue beam silk cyanobacteria belongs to (Aphanizomenon), pipe chain cyanobacteria belongs to (Aulosira), Cylindrospermopsis, cylinder spore cyanobacteria belongs to (Cylindrospermum), Loefgrenia, joint ball cyanobacteria belongs to (Nodularia), read ball cyanobacteria and belong to (Nostoc) or lumen cyanobacteria genus (Wollea).

11. methods according to claim 1, the nucleotide sequence of cyanobacteria flavodoxin of wherein encoding is SEQ ID NO.1.

12. methods according to claim 1, the nucleotide sequence of the cyanobacteria FNR that wherein encodes is SEQ ID NO.3.

13. methods according to claim 1, wherein said nucleic acid construct also comprises adjustment sequence.

14. methods according to claim 1, wherein said construct also comprises chloroplast targeted sequence.

15. methods according to claim 14, wherein said chloroplast targeted sequence derives from pea FNR.

16. methods according to claim 15, the nucleotide sequence of flavodoxin polypeptide of wherein encoding is SEQ ID NO.2.

17. methods according to claim 16, the nucleotide sequence of FNR polypeptide of wherein encoding is SEQ ID NO.4.

18. methods according to claim 1, wherein said plant is crop plants.

19. methods according to claim 18, wherein said plant is tobacco or barley.

20. methods according to claim 1, wherein said oxidative stress is caused by biological or abiotic stress.

21. methods according to claim 20, wherein said biology or abiotic stress are selected from UV-light, extreme temperature, olighydria, salinity, arid and pathogenic infection.

22. 1 kinds of nucleic acid constructs, described nucleic acid construct comprises the nucleotide sequence of the nucleotide sequence of the flavodoxin polypeptide of encoding bacterial and ferredoxin NADP (H) the reductase enzyme polypeptide of coding cyanobacteria, the nucleotide sequence of ferredoxin NADP (H) the reductase enzyme polypeptide of described coding cyanobacteria comprises the sequence that coding C holds two domain region, but does not comprise the sequence of coding phycobilisome binding domains.

23. constructs according to claim 22, wherein said bacterium is cyanobacteria.

24. constructs any one of claim 22 to 23, wherein said construct also comprises chloroplast targeted sequence.

25. constructs according to claim 24, wherein said chloroplast targeted sequence derives from pea FNR.

26. constructs according to claim 25, the nucleotide sequence of flavodoxin polypeptide of wherein encoding comprises SEQ ID NO.2.

27. according to the construct of claim 25 or 26, and the nucleotide sequence of FNR polypeptide of wherein encoding comprises SEQ ID NO.4.

28. carriers comprising the construct any one of claim 22 to 27.

29. host cells comprising the carrier of construct any one of claim 22 to 27 or claim 28.

30. unifacial leaf or the dicotyledonous plant cells comprising the carrier of construct any one of claim 23 to 27 or claim 28.

The vector of 31. constructs any one of claim 23 to 27 or claim 28 or the unifacial leaf that can be obtained by the method any one of claim 1 to 21 or dicotyledons part or vegetable cell, wherein said plant or its part comprise coding as in claim 22 to 28 the recombinant nucleic acid of polypeptide that defines.

32. according to the plant part of claim 31 or vegetable cell, and wherein said plant is crop plants.

33. according to the plant part of claim 32 or vegetable cell, and wherein said plant is tobacco or barley.

34. plant parts any one of claim 31 to 33 or the purposes of vegetable cell in the method for plant producing stress tolerance oxidative stress to reinforcement.

35. for reducing the method that in unifacial leaf or dicotyledons, ROS measures in the response of coercing, described method is included in plant the nucleic acid of the nucleic acid of the flavodoxin polypeptide of expressing encoding bacterial and ferredoxin NADP (H) the reductase enzyme polypeptide of coding cyanobacteria, the nucleic acid of ferredoxin NADP (H) the reductase enzyme polypeptide of described coding cyanobacteria comprises the sequence that coding C holds two domain region, but does not comprise the sequence of coding phycobilisome binding domains.

36., for the production of the method for product, said method comprising the steps of: plant plant any one of claim 31 to 33 and from or by the part of plant of the present invention or these plants, comprise seed, produce described product.

37. for strengthening unifacial leaf or the stress response of dicotyledons to oxidative stress or the method for patience, described method is included in plant the nucleotide sequence of the nucleotide sequence of the flavodoxin polypeptide of expressing encoding bacterial and ferredoxin NADP (H) the reductase enzyme polypeptide of coding cyanobacteria, the nucleotide sequence of ferredoxin NADP (H) the reductase enzyme polypeptide of described coding cyanobacteria comprises the sequence that coding C holds two domain region, but does not comprise the sequence of coding phycobilisome binding domains.